Abstract
Early life environmental factors are able to influence prenatal development and may cause structural and functional effects on hypothalamic–pituitary–adrenal (HPA) axis and neurotransmitter systems in the offspring. These effects seem to be species specific and may depend on the period of gestation when the factors are effective. Elevated maternal cortisol levels are assumed to play a crucial role as a programming factor during prenatal development. Thus, the present study was performed in order to examine the effects of increased maternal cortisol levels during mid- and late gestation on central and peripheral alterations of the HPA axis and brain neurotransmitter profiles in piglets. Endogenous cortisol release was induced by i.m. administration of ACTH to sows every second day either during mid- (day 49 until 75) or late gestation (day 85 until 107). Controls received injections of saline. ACTH treatment of sows during mid- and late gestation had no effects on the gestation length, the number of total born and the frequency of stillborn piglets. However, ACTH treatment during late gestation caused an increase of birth weight (P < 0.04) and affected the organ:body weight ratios (brain and adrenal) in the offspring. There was an impact of increased maternal cortisol on the HPA axis and on central neurotransmitter systems in the offspring. ACTH treatment during mid gestation caused a significant decrease of plasma corticosteroid-binding globulin (CBG; P < 0.03) and an increase of the noradrenergic activity in the locus coeruleus (LC) region (P < 0.02). Elevated maternal cortisol during late gestation also produced a significant decrease of plasma CBG (P < 0.05), but significantly increased the plasma noradrenaline (NA) concentration (P < 0.02) and decreased the serotonergic activity in the LC at both postnatal day 1 (P < 0.016) and day 28 (P < 0.003). Furthermore, there were sex-specific effects of ACTH treatment on plasma CBG, NA and brain monoamine turnover, with more pronounced changes in male offspring. In conclusion, elevated maternal cortisol levels during mid- and late gestation in pigs affect growth, HPA axis and brain neurotransmitter systems in the offspring in a sex-specific manner. The observed alterations in endocrine and neurotransmitter systems are dependent on the gestational period. Late gestation appears to be a more sensitive phase for cortisol-induced programming in pigs. Moreover, the present data show that there are marked developmental differences between laboratory animals and domestic pigs, and highlight the importance of species-specific studies on prenatal influences.
Introduction
Numerous studies show that stressors acting upon the organism during pregnancy can have distinct and long-lasting effects on the offspring. A variety of functions and systems can be influenced including behaviour, reproduction, and neuroendocrine, and autonomic and immune system (Ward 1972, Herrenkohl 1979, Weinstock et al. 1992, Maccari et al. 1995, Kay et al. 1998). Environmental factors acting early in life to organize or alter permanently these physiological systems result in ‘prenatal programming’ (Barker et al. 1993, Edwards et al. 1993). The brain is very sensitive to prenatal programming and glucocorticoids, in particular, have strong brain-programming properties. It was shown that maternal glucocorticoids may underlie the association between low birth weight and adult stress-related cardiovascular, metabolic and neuroendocrine disorders such as hypertension, type 2 diabetes, ischaemic heart disease and affective disorders (Seckl 1998). Substantial evidence suggests that prenatal stress programmes the hypothalamic–pituitary–adrenal (HPA) axis, and that plasticity of developing brain monoamine systems underlie, in part, these changes (Slotkin et al. 1992, Muneoka et al. 1997, Weinstock et al. 1998). Since an important characteristic of the stress response is the secretion of high levels of glucocorticoids, this steroid has become a primary candidate for the role of a programming factor in the prenatal stress paradigm (Phillips et al. 1998, Levitt et al. 2000, Maccari et al. 2003).
Most of the studies on prenatal stress have been performed on rodents (for a review see Braastad 1998, Matthews 2002), while very few have been performed on farm species. In cattle, transport stress during gestation resulted in offspring with increased pituitary weights and higher post-restraint cortisol concentrations (Lay et al. 1997a,b). Repeated transport stress in the last third of gestation of goats had no effect on basal cortisol concentrations in goat kids, but influenced the sympatho-adrenomedullary system (Roussel et al. 2005). Isolation stress during gestation in sheep caused higher basal cortisol concentrations in the offspring (Roussel et al. 2004) and it was also demonstrated in sheep that prenatal exposure to synthetic glucocorticoids elevated the basal cortisol levels and increased the cortisol response to corticotrophin-releasing hormone plus arginine vasopressin challenge later in life (Moss et al. 2001, Sloboda et al. 2002). In pigs, stimulation of maternal cortisol release by weekly maternal adrenocorticotrophic hormone (ACTH) injections and restraint in pregnant sows for 6 weeks resulted in offspring with higher adrenocortical expression of ACTH receptor mRNA and a higher adrenal cortex:medulla area ratio on day 1 of age indicating greater adrenal sensitivity. Under stressful conditions, the pigs in the maternal ACTH-treated group also showed significantly higher plasma cortisol responses (Haussmann et al. 2000). Recent studies in pigs have also shown alterations in the offspring’s HPA axis activity after hydrocortisone treatment or social stress during gestation dependent on the timing of exposure (Jarvis et al. 2006, Kranendonk et al. 2006). These alterations may be related to programming of brain corticosteroid receptors. In a previous study of our group, we could show that piglets, whose mothers were subjected to daily restraint stress for the last 5 weeks of gestation, exhibit reduced hypothalamic glucocorticosteroid receptor (GR)-binding sites, but increased hippocampal GR sites with no effect on the hippocampal mineralocorticoid receptor (MR), possibly indicating decreased negative feedback at the paraventricular nucleus (PVN) and enhanced facilitation of the HPA response (Kanitz et al. 2003).
The stressors used in most prenatal stress studies may also activate other stress systems, e.g. neurotransmitters and opioids, so that it is unlikely that the observed changes in the offspring are caused by the action of maternal glucocorticoids alone. In order to determine whether elevated maternal glucocorticoids are responsible for the observed effects in postnatal pigs, we developed a model of repeated administration of exogenous ACTH to pregnant sows that stimulates the endogenous maternal cortisol response. It has already been shown that repeated intramuscular administration of ACTH to pregnant sows results in a prolonged elevation of their plasma cortisol levels and that this maternal adrenocortical stimulation increases the fetal cortisol levels, which may affect the development in utero (Otten et al. 2004, Schwerin et al. 2005).
Little is known about the sensitivity of the growing fetus to programming effects of maternal glucocorticoids during specific periods of gestation. The timing of maturation of the HPA axis relative to birth is highly species specific. In animals that give birth to mature young (e.g. guinea pigs, sheep and primates), maximal brain growth and a large proportion of neuroendocrine maturation (including corticosteroid receptor development) take place in utero (Matthews 1998, Challis et al. 2000). Conversely, in species that give birth to immature young (e.g. rats, rabbits and mice), much of the brain and neuroendocrine development occurs in the postnatal period (Dentet al. 2000). Therefore, manipulations of the maternal environment and/or hormonal milieu in gestation will influence different stages of brain and HPA development depending on the species. The marked developmental differences between rodents and farm animals highlight the value of comparative studies on this topic. Moreover, pigs show a different placental structure compared with rats with a placentation characterized as epitheliochorial, whereas that of the rodent is haemoendothelial. Therefore, the pig placenta may offer a more significant barrier to maternal hormonal influence.
The aim of this study was to examine the effects of increased maternal glucocorticoid levels on central and peripheral alterations of the HPA axis and brain neurotransmitter profiles in neonatal pigs. The possible different impact of increased maternal glucocorticoid levels at specific prenatal ontogenetic periods was studied by incorporating mid-gestation as a period of ongoing HPA axis maturation and fetal-growth acceleration (Klemcke & Christenson 1997, McPherson et al. 2004), and late gestation as the period of major brain growth spurt in pigs (Pond et al. 2000).
Materials and Methods
Animals and housing
Forty-four pregnant primiparous Landrace sows and their litters were used in two experiments (EXP 1, n = 24; EXP 2, n = 20). Sows were individually housed in pens (1.2 × 2.0 m), and fed twice a day with a commercial pig diet for pregnant sows. The ration increased from 2.2 kg/day in week 8 of gestation to 3.2 kg/day in the last week of gestation and was totally consumed by the animals. Water was available ad libitum. Approximately, 1 week before farrowing, at day 105 of gestation, sows were weighed (EXP 1, 202.2 ± 2.2 kg; EXP 2, 223.3 ± 3.1 kg) and moved to individual farrowing pens (2.0 × 3.0 m). There were no differences in the body weight of sows between the treatment groups within each experiment. Sows stayed in the farrowing pens with their offspring until weaning on day 28 and during this period, piglets were weighed at day 1, 14 and 28 of age. In both the experiments, no abortions occurred.
All the procedures involving animal handling and treatment were approved by the Committee for Animal Use and Care of the Ministerial Agricultural Department of Mecklenburg-Vorpommern, Germany.
Experimental procedure
In EXP 1 (mid-gestation, MG), 12 sows received i.m. administrations of synthetic ACTH (Synacthen Depot, Novartis Pharma, Brussels, Belgium) at a dose of 100 IU per animal every 2 days (MG-ACTH), beginning on day 49 until day 75 of gestation. Administrations of ACTH were given to the unrestrained sows in the neck at 0800 h and injection sites within the neck region were changed every treatment day. The other 12 sows served as a control (MG-C) and received saline repeatedly. We have already shown in a previous study that this ACTH administration protocol during mid-gestation results in a consistent cortisol response throughout the treatment period with increased cortisol levels until 8 h post-injection (Otten et al. 2004).
In EXP 2 (late gestation, LG), ten sows were repeatedly injected with 100 IU synthetic ACTH (LG-ACTH) as described earlier (every 2 days), starting on day 85 until day 107 of gestation, whereas the other ten sows served as a control treated with saline (LG-C). During late gestation, ACTH application also caused a consistent cortisol enhancement throughout the treatment period (W Otten, unpublished observations).
In both EXP 1 and EXP 2, litters were standardized to ten animals after birth, usually five males and five females. Surplus animals were fostered to other sows in the pig unit. Blood samples were taken on postnatal day (PND) 1 and day 28 from all the piglets by anterior vena cava puncture in a supine position (the whole procedure lasted approximately 30 s). For plasma extraction, blood samples were collected in ice-cooled polypropylene sampling tubes containing EDTA (Kabevette, Kabe Labortechnik GmbH, Nürnbrecht-Elsenroth, Germany), immediately placed on ice and subsequently centrifuged at 2000 g for 15 min at 4 °C. Plasma was stored at −80 °C until hormone analysis.
Tissue collection
On PND1 and PND28, two male and two female piglets from each litter were euthanized by an i.v. injection of T61 (embutramide/mebezonium iodide/tetracaine hydrochloride, Intervet, Unterschleissheim, Germany). The brains were quickly removed and placed on ice, and the hippocampus, amygdala, hypothalamus including the PVN, and the region of locus coeruleus (LC) were dissected, frozen in liquid nitrogen, and stored at −80 °C. The stereotaxic atlas of the pig brain (Felix et al. 1999) served as reference. Both adrenal glands were also removed, weighed and frozen in liquid nitrogen.
All the experimental procedures on the animals were performed between 0800 and 1100 h.
Catecholamine analyses
Plasma concentrations of adrenaline (A) and noradrenaline (NA) were analysed in duplicate using high-pressure liquid chromatography (HPLC) with electrochemical detection after extraction from plasma samples by absorption on aluminium oxide (Otten et al. 1997). Intra- and inter-assay coefficients of variation (CV) were 11.5 and 12.4% for adrenaline, and 2.1 and 1.9% for noradrenaline.
Cortisol analysis
Plasma cortisol concentrations were measured in duplicate using a commercially available 125I-RIA kit (DRG International Inc., Mountainside, NJ, USA) according to the manufacturer’s guidelines. Cross-reactivities of antibody used to any potentially competing plasma steroids (DRG International Inc.) were lower than 5%. The assay was validated for use with porcine plasma. Sensitivity of the assay was 3 ng/ml, and intra- and inter-assay CV values were 5.3 and 9.8% respectively.
Corticosteroid-binding globulin (CBG)
Plasma samples were examined for CBG using a modified binding assay previously described by Kanitz et al.(2002). Briefly, 25 μl blood plasma were incubated with 0.78 nM unlabelled cortisol (hydrocortisone, Merck) and 25 pM [3H]-cortisol (specific radioactivity 68 Ci/mmol, Amersham Pharmacia Biotech). Non-specific binding was determined in parallel using a 100-fold excess of cold cortisol. The separation of bound and free [3H]-cortisol was performed by precipitation with dextran-coated charcoal at 4 °C and subsequent centrifugation at 1000 g for 10 min. The intra-and inter-assay CV values were 7.8 and 9.1% respectively.
Glucocorticosteroid receptor (GR)-binding assay
The GR-binding assay was conducted as previously described by Kanitz et al.(1998). Briefly, tissues of hippocampus and hypothalamus were homogenized in ice-cold 10 mM Tris–HCl, pH 7.5, containing 12.5 mM EDTA, 10 mM sodium molybdate, 0.25 mM saccarose and 1 mM dithiothreitol using a Teflon-glass homogenizer. The homogenate was centrifuged at 120 000 g for 60 min at 0–4 °C to obtain cytosol (i.e. the supernatant fraction).
Hippocampal GR binding was evaluated directly in saturation experiments using the pure glucocorticoid [3H]-dexamethasone (specific activity 43 Ci/mmol; Amersham Pharmacia Biotech) over a concentration range of 0.2–24 nM. For the single-point assay of the GR binding in the hypothalamus, a 100 μl aliquot of cytosol was incubated with 5 μl aliquots of a saturating 10 nM concentration of [3H]-dexamethasone. In both single-point and saturation experiments, non-specific binding was determined with a parallel incubation that contained 500-fold excess of RU 28362 (kindly donated by Roussel Uclaf, Romainville, France), which binds selectively to the glucocorticoid receptor. The separation of bound from free ligand was performed by precipitation with dextran-coated charcoal and the receptor–[3H]-steroid complexes were counted in a spectral liquid scintillation counter (LKB Wallac, Turku, Finland) at an efficiency of 50%.
The theoretical maximal number of binding sites (Bmax) and Kd for [3H]-dexamethasone binding in hippocampus were derived from saturation experiments using the method of Scatchard (1949). The results of the single-point assays were expressed in terms of specific binding per milligram protein. Protein content was determined by the method of Lowry et al.(1951) with BSA as standard. Data were expressed as femtomole per milligram of protein.
Quantification of monoamines and metabolites
NA, its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine (DA), its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the LC were determined using HPLC and electrochemical detection. Samples were weighed and homogenized on ice for 5 min with a hand homogenizer in 0.5 ml of 0.2 M perchloric acid followed by centrifugation at 45 000 g for 10 min at 4 °C. After collection of the supernatants, the procedure was repeated. Pooled supernatants of the repeated extractions were again centrifuged at 45 000 g for 10 min at 4 °C. Samples of 20 μl were then injected directly in the HPLC system equipped with a 125 × 4 mm reversed-phase column packed with Prontosil C18 AQ (Bischoff Analysentechnik, Leonberg, Germany). As mobile phase, 58 mM sodium hydrogen phosphate buffer containing 1.2 mM octansulphonic acid, 0.3 mM EDTA, 0.2 mM potassium chloride and 6% methanol at pH 3.5 was used at a flow rate of 0.8 ml/min. Electrochemical detection was achieved by an ISAAC in situ Ag/AgCl cell with a glassy carbon working electrode set at a potential of 600 mV (Shimadzu, Duisburg, Germany). Dihydroxybenzylamine was used as an internal standard for quantification and the concentrations were expressed as picogram per milligram weight tissue. As an index of NA, DA and 5-HT turnover, the MHPG/NA, DOPAC/DA, HVA/DA and 5-HIAA/5-HT ratios were calculated.
Adrenal glands
For histological analyses, the right adrenal gland was sectioned (7 μm) with a cryostat microtome (Reichert-Jung, Leica, Nussloch, Germany), transverse to the long axis, in the mid-glandular region. Sections were stained using haematoxylin and eosin dye for visualization of both the adrenal cortex and the medulla (Fiedler et al. 1996). Using a video camera (CF15/2RGB, KAPPA, Gleichen, Germany) and a computer system, images of three sections were recorded. The adrenal capsule, the line of demarcation between adrenal cortex and medulla as well as any open space where a blood vessel resided, were traced. The CV for the area of the three sections within the same gland was 1.6% for the adrenal cortex and 1.9% for the adrenal medulla. The tracings were then used to quantify the area of the entire gland, the cortex and the medulla (excluding open space area) using an image analysis system (SIS, Münster, Germany). For each selected section, the number of labelled cells was counted and the data were expressed as densities (number of cells/mm2).
Statistical analysis
Data analyses were performed using the MIXED model procedure of SAS (Statistical Analysis System Institute 1999). Within each experiment, the following analyses were carried out: (1) body weights, relative tissue weights, glucocorticoid receptor binding, and biogenic amine contents in brain tissues were analysed with a model including the fixed factors treatment and gender, the corresponding interaction and the random factor sow nested within treatment; (2) plasma cortisol, CBG and catecholamines, area and number of cells in adrenal tissues were analysed using the same model with the body weight as an additional covariate. Tukey post hoc tests were used, where appropriate, to explore the source of significant effects. Significance was considered at P < 0.05 and tendencies at P < 0.10. All data are presented as least square mean ± s.e.m.
Results
Gestation outcome, body and organ weights of the offspring
ACTH treatment of sows during mid- and late gestation had no effect on the gestation length (MG-ACTH: 114.8 ± 0.3 days, MG-C: 115.2 ± 0.3 days; LG-ACTH: 115.1 ± 0.4 days, LG-C: 115.4 ± 0.4 days) nor on the number of total born piglets (MG-ACTH: 12.3 ± 1.0, MG-C: 12.2 ± 1.0; LG-ACTH: 13.6 ± 0.8, LG-C: 13.2 ± 0.9). There was also no significant difference in the frequency of stillborn piglets during both the treatment periods (MG-ACTH: 2.9 ± 1.5%, MG-C: 5.0 ± 2.1%; LG-ACTH: 5.2 ± 2.1%, LG-C: 4.4 ± 2.1%).
Birth and body weights of MG-ACTH piglets did not differ from control piglets and there were no differences in brain and adrenal gland weights expressed as organ:body weight ratio (Table 1). In contrast, ACTH treatment during late gestation significantly increased the birth weight in the offspring (P < 0.041) and the body weight on PND14 (P < 0.019; Table 1). Furthermore, analysis revealed a significant decrease in brain:body weight ratio on PND1 (P < 0.049) and an increase in adrenal:body weight ratio on PND28 (right, P < 0.05; left, P < 0.10). While the decrease in brain:body weight ratio was caused by an increase in body weight on PND1, the increase in adrenal:body weight ratio on PND28 corresponded to a significant increase in adrenal weights of the LG-ACTH piglets (right (LG-ACTH): 0.488 ± 0.010 g, LG-C: 0.418 ± 0.012 g, P < 0.001; left (LG-ACTH): 0.493 ± 0.012 g, LG-C: 0.433 ± 0.014 g, P < 0.008).
Plasma cortisol, CBG and catecholamines
ACTH stimulation of sows during mid- and late gestation did not affect the basal cortisol levels in the offspring either on PND1 or on PND28 (P > 0.1; Fig. 1A), but the statistical analyses revealed a significant influence of ACTH treatment on CBG concentrations on PND1. As shown in Fig. 1B, CBG concentrations were decreased in both MG-ACTH offspring (P < 0.034) and LG-ACTH offspring (P < 0.049) on PND1. In addition, a significant sex × prenatal treatment effect (P < 0.005) on CBG levels was observed on PND28 in LG-ACTH piglets. Post hoc testing revealed that male piglets from ACTH sows had significantly higher CBG concentration (P < 0.009) compared to male controls.
Plasma NA concentrations after venipuncture were not affected in MG-ACTH piglets (P > 0.05), but were significantly increased in LG-ACTH piglets on PND1 (P < 0.016; Fig. 2A). Although there were no main effects, either of sex or of prenatal treatment after ACTH application during mid-gestation, there was a significant sex × prenatal treatment interaction effect on plasma NA levels of MG-ACTH piglets on PND28 (P < 0.0104). Post hoc testing revealed that male piglets from ACTH sows displayed higher NA values (P < 0.035) compared with male control piglets at this age.
Plasma adrenaline concentrations of piglets were not affected by ACTH stimulation of their mothers during either of the treatment periods (Fig. 2B).
Brain monoamines and metabolites
A summaryof monoamine and metabolite levels in LC region of MG-ACTH piglets at different ages is presented in Table 2. Both sex and prenatal treatment altered monoamine and metabolite levels in the LC of MG-ACTH piglets. There was no significant main effect of prenatal treatment on monoamine and metabolite levels in piglets on PND1. However, a significant sex × prenatal treatment interaction effect on MHPG level was observed (P < 0.049), where MHPG concentrations increased in male MG-ACTH piglets and decreased in female MG-ACTH piglets. The 5-HT concentration (P < 0.003) and the 5-HT metabolite 5-HIAA (P < 0.018) were elevated in males, regardless of prenatal treatment, compared with females. As shown in Table 2, on PND28, a significant main effect of prenatal treatment was found on MHPG (P < 0.023) and in tendency on NA (P < 0.10) and HVA (P < 0.066). Additionally, there were significant effects of sex on NA (P < 0.039) and DA (P < 0.05), with higher concentrations observed in female MG-ACTH piglets.
The effects of ACTH treatment during late gestation on monoamines and metabolites in the LC of piglets are shown in Table 3. There was also an influence of both sex and prenatal treatment on the neurochemical activity in the offspring. ACTH treatment of the mothers significantly decreased the 5-HT levels in the offspring on both PND1 (P < 0.016) and PND28 (P < 0.003) and decreased, in tendency, the 5-HIAA levels on PND1 (P < 0.066). Additionally, there was a significant effect of sex on 5-HT levels in the LC on PND1 (P < 0.05), with higher levels observed in males compared with females. A significant sex × prenatal treatment interaction effect on 5-HIAA/5-HT ratio was observed on PND1, where values decreased in male LG-ACTH piglets and increased in female LG-ACTH piglets (P < 0.019). Furthermore, the DOPAC/DA ratio was significantly affected by sex on PND1 (P < 0.03), female LG-ACTH piglets displayed higher values compared with male animals. On PND28, there was a main effect of prenatal treatment on HVA/DA ratio (P < 0.05), piglets from ACTH-treated sows displayed higher values (Table 3).
Glucocorticoid receptor binding in hypothalamus and hippocampus
There was a tendency for reduced GR binding in the hypothalamus of newborn piglets (PND1) from both treatments compared with controls (MG-ACTH, P < 0.068; LG-ACTH, P < 0.079; Fig. 3A). The hippocampal GR binding was not affected by treatment either during mid- or during late gestation (Fig. 3B). There were no effects of sex on the GR binding in these brain areas.
Adrenal glands
ACTH-treatment of sows during mid gestation did not induce morphological alterations of the adrenal gland in the offspring. However, ACTH-treatment during late gestation significantly increased the area of adrenal cortex in piglets on PND28 (P < 0.037; Fig. 4A) and increased the density of cells in the adrenal medulla on PND1 (P < 0.02; Fig. 5B).
Discussion
The present study indicates that elevated maternal cortisol exposure during gestation in pigs affects growth, HPA axis at different levels and brain neurotransmitter profiles in the offspring in a sex-specific manner. The effects on endocrine and neurochemical key parameters depend on the gestational stage, and the period of late gestation appears to be a particularly vulnerable developmental stage during porcine brain differentiation.
Administration of ACTH to pregnant sows during mid-and late gestation had no effect on the gestation length, the number of total born and the frequency of stillborn piglets. This is in accordance with previous studies in pigs using different stressors such as maternal restraint, ACTH injections or treatments with hydrocortisone acetate (Haussmann et al. 2000, Otten et al. 2001, Kranendonk et al. 2005). However, elevated maternal glucocorticoids during late gestation caused an increase in birth weight of LG-ACTH piglets. Such higher birth weights after prenatal ACTH or stress treatments were described for calves and lambs (Lay et al. 1997b, Roussel et al. 2004), whereas in rodents and humans, maternal restraint stress or prenatal dexamethasone treatment generally decreases the birth weights (Bloom et al. 2001, Lesage et al. 2004, Burlet et al. 2005). This discrepancy may result from differences in the fetal development as well as species sensitivity to glucocorticoids.
Maternal elevated cortisol during late gestation also affected brain and adrenal weights in the offspring. The effects resulted in a reduction of the brain:body weight ratio on PND1, which was due to the increased body weight of LG-ACTH piglets at birth. On PND28, adrenal:body weight ratio and adrenal weights were increased in LG-ACTH piglets. Similar results were found in guinea pigs after prenatal dexamethasone treatment (Liu et al. 2001, Banjanin et al. 2004). Our results also revealed an increased area of the adrenal cortex in these animals, indicating that internal morphological changes occurred, which increased the cortical mass. This is in accordance with the results from a previous study in pigs, where prenatal ACTH treatment and restraint caused an increased adrenal cortex:medulla ratio in the offspring (Haussmann et al. 2000). These findings indicate that the adrenal may have a higher capacity for cortisol release under stressful conditions as already shown in prenatally stressed pigs (Haussmann et al. 2000, Jarvis et al. 2006).
HPA activity is regulated by a negative feedback process in which circulating glucocorticoids act at various target sites in the brain and pituitary. CBG influences the bioavailability as well as metabolic clearance rate of cortisol within the circulation (Bright 1995). The amount of cortisol, which is taken up by target tissue, is approximated by the non-CBG bound (free + albumin) fraction of total circulating steroid (Siiteri et al. 1982). Thus, differences in circulating CBG levels determine the magnitude of the glucocorticoid negative feedback signal (Walker et al. 1990). In the present study, elevated maternal cortisol during both the mid- and late gestation period significantly decreased plasma CBG concentrations in piglets on PND1, whereas total plasma cortisol concentrations were not changed. A stress-associated decline in plasma CBG concentrations was found in rats (Armario et al. 1994, Spencer et al. 1996), horses (Alexander & Irvine 1998) and pigs (Heo et al. 2003). It has not been demonstrated directly whether stress-related changes in plasma CBG result from changes in production, metabolic degradation and/or transfer to target tissues. However, Heo et al.(2005) found in fetal pigs a positive correlation between plasma CBG concentrations and hepatitic CBG mRNA. Therefore, it is assumed that the decrease of CBG levels in newborn piglets may result from direct inhibition of fetal hepatitic CBG biosynthesis by elevated maternal cortisol exposure during both mid- and late gestation. This decrease in CBG and the unchanged total cortisol levels indicate a higher amount of biologically available free cortisol, which is also found in animals under conditions of chronic stress (Kim et al. 1999, Stefanski 2000, Heo et al. 2005).
Glucocorticoids regulate the HPA axis at the level of the pituitary, the hypothalamus, and the supra-hypothalamic limbic structures, particularly the hippocampus and amygdala. As maternal glucocorticoids cross the placenta, albeit with partial degradation by 11β-hydroxysteroid-dehydrogenase, they could affect the maturation of the fetal HPA axis, determining the set-point for feedback. In the present study, elevated maternal cortisol during both mid- and late gestation caused a tendency for reduced hypothalamic GR binding, but did not alter the GR binding in the hippocampus on PND1. In rats, prenatal glucocorticoid exposure permanently reduced the hippocampal GRs and increased basal corticosterone levels, changes thought to attenuate HPA axis feedback sensitivity in anticipation of stress (Levitt et al. 1996, Welberg et al. 2001). Unlike rats, injections of dexamethasone during late gestation in guinea pigs increased the GRs in the hippocampus (Dean & Matthews 1999). An increase in hippocampal GRs and a decrease in the number of GRs in the hypothalamus were also found in pigs prenatally stressed by repeated restraint during late gestation (Kanitz et al. 2003). The results of the present study demonstrate that elevated maternal cortisol during both mid- and late gestation periods acts at the level of the hypothalamus by decreasing the GR-binding capacity and may therefore alter the set-point and attenuate the HPA axis feedback. A possible reason for the different results between the brain areas could be their heterochronous development. It has been shown in guinea pigs, a species that, similar to the domestic pig and in contrast to rats, delivers mature young, GRs develop earlier in the fetal hypothalamic PVN than in hippocampus. While hippocampal GRs reach their highest concentrations near term, the level in the PVN is highest at the beginning of the third trimester, and decreases dramatically in the last 3 weeks (Matthews 1998). A similar reduction in GR mRNA in the PVN has also been noted in the fetal sheep near term (Andrews & Matthews 2000). Although the establishment of porcine fetal HPA axis was shown around gestational day 77 (Schwerin et al. 2005), at present, data on GR development and maturation in pigs are not available. However, a related time course of brain development in pigs and guinea pigs is assumed because of similarities in fetal HPA function and brain growth spurt (transient period of growth when the brain is growing most rapidly) during late gestation in these species (Fowden et al. 1998, Pond et al. 2000). The differences in GR regulation in this study may also be due to different receptor sensitivities between these species.
Some studies in rats and also in guinea pigs indicate that programming of corticosteroid receptors, such as the HPA axis, is possibly sex-specific with the effect of prenatal glucocorticoids being larger in female offspring (Weinstock et al. 1992, Dean & Matthews 1999, Szuran et al. 2000, Bowman et al. 2004, Gerardin et al. 2005). In pigs, we found no sex-specific effects on brain GRs, but an interaction of sex and treatment on plasma CBG concentrations in 28-day-old LG-piglets. Although there was no significant effect on CBG concentration at this age, male pigs from mothers treated with ACTH during late gestation expressed higher CBG levels than male control animals. These findings could point to a sex-dependent impact of prenatal elevated cortisol on hepatitic CBG production in pigs.
Several reports indicate that prenatal stress in rats may induce an enhanced activation of the sympathetic nervous system and may affect the concentrations and turnover of brain catecholamines (Takahashi et al. 1992, Rudeen & Weinberg 1993, Muneoka et al. 1997, Weinstock et al. 1998). In pigs, maternal restraint stress during late gestation did not cause significant effects on plasma and brain catecholamine levels in the offspring (Otten et al. 2001, Kanitz et al. 2003). In contrast, the present results revealed an influence of elevated maternal cortisol on plasma NA concentrations and monoamine turnover in the LC, a key brainstem region of the noradrenergic system. While ACTH treatment during mid gestation increased MHPG and NA concentrations in the LC region of 28-day-old piglets and decreased HVA content, elevated cortisol exposure during late gestation increased NA concentrations in plasma samples taken by venipuncture on PND1. These results are partly consistent with a study in rats, where it was found that prenatal stress causes an increased turnover of NA and a reduced DA concentration in the LC (Takahashi et al. 1992). Furthermore, it was shown that increases in LC activity were correlated with arousal and a general increase in sympathetic activation (Berridge & Foote 1991). The increased levels of NA and MHPG after prenatal cortisol exposure during mid gestation may therefore reflect a higher noradrenergic activity in the offspring. In contrast, cortisol exposure during late gestation did not affect the noradrenergic activity in the LC region, but persistently decreased 5-HT concentrations on PND1 and PND28. A study with mice and rats revealed a significant decrease of 5-HT levels in the LC after repeated postnatal restraint stress (Konstandi et al. 2000). Serotonergic neurons in the LC are presumed to be involved in stress-induced disorders like depression and it was found that conditioned fear diminishes the 5-HT release in the LC, indicating that anxiety is associated with decreased serotonergic activity in the LC (Kaehler et al. 2000). It is suggested that the decrease in 5-HT levels in brain of prenatally stressed offspring may be due to a reduction of tryptophan hydroxylation (Hayashi et al. 1998). Our findings demonstrate that prenatal cortisol exposure elicits different effects on the monoamine system in the LC of the offspring with more pronounced effects on the noradrenergic activity after mid-gestational and on the serotonergic activity after late-gestational exposure. This may reflect the different susceptibility of these systems to cortisol during the ongoing prenatal brain development. Differences in these monoamine systems were detectable on PND28 and indicate that there could be long-lasting effects on the behavioural and neurophysiological response to stimuli later in life.
In summary, the present results demonstrate that elevated maternal cortisol levels during mid- and late gestation in pigs cause alterations of the HPA axis and of central neurotransmitter systems in the offspring, and in some cases, in a sex-specific manner. In general, increased cortisol exposure of fetuses during both periods leads to decreased plasma CBG concentrations, which indicate a higher amount of biologically available free cortisol. Elevated maternal cortisol during mid gestation specifically increased the noradrenergic activity in the LC, whereas serotonergic activity in the LC was decreased only after late gestational treatment. Sex-specific effects were generally more pronounced in the male offspring. In the pig, the temporal characteristics of neurotransmitters and HPA axis development are not known and, to our knowledge, we are the first group to report these cortisol-dependent alterations in porcine brain. Furthermore, the main period of sexual brain differentiation in pigs seems to be during late gestation, although sex-specific influences on the noradrenergic system were found after mid-gestational treatment.
The present data show that there are marked developmental differences between laboratory animals and domestic pigs and highlight the importance of species-specific studies on prenatal influences. Increases in maternal cortisol could be one of the mechanisms involved in the effects of prenatal stress in pigs. According to the present findings, further studies are necessary to determine long-term consequences on coping behaviour and the adaptive ability of pigs to husbandry practices.
Body weights and organ weights of brain and adrenal glands in piglets from sows treated with adrenocorticotrophic hormone (ACTH) (Synacthen Depot 100 IU i.m.) or saline during mid- and late gestation. Values are least square mean ± s.e.m. Number of animals per group: BW at birth (MG-ACTH = 120, MG-C = 120, LG-ACTH = 100, LG-C = 100), BW PND14 and PND28 (MG-ACTH = 72, MG-C = 72, LG-ACTH = 60, LG-C = 60); brain and adrenal gland PND1 and PND28 (MG-ACTH = 48, MG-C = 48, LG-ACTH = 40, LG-C = 40).
| Mid gestation | Late gestation | |||
|---|---|---|---|---|
| ACTH | Control | ACTH | Control | |
| *Within a row denotes significant differences (P < 0.05) between ACTH and controls. | ||||
| Body weight (BW) (kg) | ||||
| At birth | 1.18 ± 0.06 | 1.22 ± 0.06 | 1.34 ± 0.05* | 1.18 ± 0.05* |
| PND14 | 3.70 ± 0.27 | 3.97 ± 0.25 | 4.82 ± 0.12* | 4.33 ± 0.13* |
| PND28 | 7.09 ± 0.42 | 7.49 ± 0.38 | 8.60 ± 0.29 | 8.06 ± 0.33 |
| Brain (g/kg BW) | ||||
| PND1 | 19.64 ± 0.44 | 19.74 ± 0.47 | 18.66 ± 0.84* | 21.39 ± 0.92* |
| PND28 | 5.91 ± 0.31 | 5.69 ± 0.28 | 5.10 ± 0.18 | 5.08 ± 0.20 |
| Adrenal gland (g/kg BW) | ||||
| Left | ||||
| PND1 | 0.131 ± 0.040 | 0.128 ± 0.004 | 0.110 ± 0.005 | 0.113 ± 0.005 |
| PND28 | 0.062 ± 0.003 | 0.062 ± 0.003 | 0.058 ± 0.003 | 0.052 ± 0.003 |
| Right | ||||
| PND1 | 0.120 ± 0.004 | 0.117 ± 0.005 | 0.102 ± 0.006 | 0.102 ± 0.006 |
| PND28 | 0.65 ± 0.004 | 0.062 ± 0.004 | 0.057 ± 0.002* | 0.050 ± 0.002* |
Effects of maternal adrenocorticotrophic hormone (ACTH) (Synacthen Depot 100 IU i.m.) or saline treatment during mid gestation on LC monoamine levels and their turnover in piglets on PND1 and PND28. Values are least square mean ± s.e.m. Number of animals per group: PND1 (ACTH = 20, control = 12); PND28 (ACTH = 15, control = 20).
| PND1 | PND28 | |||
|---|---|---|---|---|
| ACTH | Control | ACTH | Control | |
| *Within a row denotes significant differences (P < 0.05) between ACTH and controls. NA, noradrenaline; MHPG, 3-methoxy-4-hydroxyphenylglycol; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindole-3-acetic acid. | ||||
| NA (pg/mg) | 1255.5 ± 103.4 | 1249.6 ± 108.7 | 1364.0 ± 72.1 | 1165.8 ± 74.0 |
| MHPG (pg/mg) | 92.7 ± 12.3 | 90.9 ± 12.5 | 92.2 ± 4.7* | 71.2 ± 5.1* |
| MHPG/NA | 0.08 ± 0.01 | 0.08 ± 0.02 | 0.07 ± 0.01 | 0.07 ± 0.01 |
| DA (pg/mg) | 172.8 ± 26.4 | 168.1 ± 27.6 | 151.5 ± 20.8 | 122.7 ± 21.3 |
| DOPAC (pg/mg) | 353.9 ± 28.6 | 281.5 ± 37.8 | 188.4 ± 23.5 | 220.4 ± 21.5 |
| HVA (pg/mg) | 688.4 ± 51.8 | 602.5 ± 66.4 | 209.3 ± 39.3 | 331.0 ± 34.0 |
| DOPAC/DA | 1.97 ± 0.14 | 1.76 ± 0.18 | 1.31 ± 0.19 | 1.52 ± 0.31 |
| HVA/DA | 4.03 ± 0.24 | 4.16 ± 0.30 | 1.71 ± 0.39 | 2.65 ± 0.34 |
| 5-HT (pg/mg) | 1001.9 ± 123.6 | 794.9 ± 146.3 | 1052.2 ± 71.1 | 1093.7 ± 67.8 |
| 5-HIAA (pg/mg) | 1826.8 ± 290.3 | 1417.8 ± 343.9 | 790.6 ± 79.8 | 840.8 ± 79.3 |
| 5-HIAA/5-HT | 1.89 ± 0.09 | 1.79 ± 0.12 | 0.74 ± 0.04 | 0.78 ± 0.04 |
Effects of maternal adrenocorticotrophic hormone (ACTH) (Synacthen Depot 100 IU i.m.) or saline treatment during late gestation on locus coeruleus (LC) monoamine levels and their turnover in piglets on PND1 and PND28. Values are least square mean ± s.e.m. Number of animals per group: PND1 (ACTH = 27, control = 22); PND28 (ACTH = 40, control = 32).
| PND1 | PND28 | |||
|---|---|---|---|---|
| ACTH | Control | ACTH | Control | |
| *Within a row denotes significant differences (P < 0.05) between ACTH and controls. †Within a row denotes significant differences (P < 0.01) between ACTH and controls. NA, noradrenaline; MHPG, 3-methoxy-4-hydroxyphenylglycol; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindole-3-acetic acid. | ||||
| NA (pg/mg) | 1030.4 ± 75.3 | 1203.3 ± 77.1 | 1236.0 ± 98.4 | 1397.3 ± 112 |
| MHPG (pg/mg) | 120.7 ± 38.7 | 91.4 ± 45.6 | 68.5 ± 16.0 | 102.0 ± 19.1 |
| MHPG/NA | 0.12 ± 0.03 | 0.10 ± 0.03 | 0.07 ± 0.02 | 0.08 ± 0.02 |
| DA (pg/mg) | 163.9 ± 20.1 | 164.1 ± 21.0 | 137.9 ± 8.0 | 151.9 ± 9.1 |
| DOPAC (pg/mg) | 314.7 ± 38.9 | 330.7 ± 41.5 | 251.8 ± 32.8 | 196.3 ± 32.7 |
| HVA (pg/mg) | 420.4 ± 47.0 | 428.9 ± 48.7 | 378.5 ± 43.5 | 293.1 ± 49.2 |
| DOPAC/DA | 1.64 ± 0.21 | 1.99 ± 0.22 | 1.40 ± 0.09 | 1.24 ± 0.08 |
| HVA/DA | 2.74 ± 0.30 | 2.85 ± 0.33 | 2.57 ± 0.19* | 1.93 ± 0.22* |
| 5-HT (pg/mg) | 828.1 ± 58.8* | 1071.2 ± 63.7* | 959.0 ± 52.1† | 1258.3 ± 58.9† |
| 5-HIAA (pg/mg) | 1276.0 ± 168.1 | 1782.1 ± 185.3 | 772.8 ± 62.2 | 960.5 ± 70.3 |
| 5-HIAA/5-HT | 1.59 ± 0.10 | 1.68 ± 0.11 | 0.84 ± 0.04 | 0.77 ± 0.05 |
(A) Basal plasma cortisol and (B) CBG concentrations in piglets on PND1 and PND28 from sows treated with ACTH (Synacthen Depot 100 IU i.m.; filled bars) or saline (open bars) during mid- and late gestation. *P < 0.05. Number of animals per group: cortisol, PND1 (MG-ACTH = 88, MG-C = 94, LG-ACTH = 100, LG-C = 95), PND28 (MG-ACTH = 37, MG-C = 46, LG-ACTH = 60, LG-C = 50); CBG, PND1 (MG-ACTH = 103, MG-C = 103, LG-ACTH = 100, LG-C = 94), PND28 (MG-ACTH = 37, MG-C = 46, LG-ACTH = 59, LG-C = 50).
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06868
(A) Plasma noradrenaline and (B) adrenaline concentrations in piglets on PND1 and PND28 from sows treated with ACTH (Synacthen Depot 100 IU i.m.; filled bars) or saline (open bars) during mid- and late gestation. *P < 0.05. Number of animals per group: noradrenaline, PND1 (MG-ACTH = 102, MG-C = 101, LG-ACTH = 100, LG-C = 96), PND28 (MG-ACTH = 37, MG-C = 40, LG-ACTH = 59, LG-C = 51); adrenaline, PND1 (MG-ACTH = 102, MG-C = 101, LG-ACTH = 100, LG-C = 96), PND28 (MG-ACTH = 37, MG-C = 49, LG-ACTH = 59, LG-C = 51).
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06868
Glucocorticoid receptor (GR) binding in (A) hypothalamus and (B) hippocampus of piglets on PND1 and PND28 from sows treated with ACTH (Synacthen Depot 100 IU i.m.; filled bars) or saline (open bars) during mid- and late gestation. There is a tendency for reduced GR binding in the hypothalamus on PND1 during both mid- (P < 0.07) and late (P < 0.08) gestation. Number of animals per group: hypothalamus, PND1 (MG-ACTH = 22, MG-C = 21, LG-ACTH = 25, LG-C = 21), PND28 (MG-ACTH = 17, MG-C = 20, LG-ACTH = 37, LG-C = 32); hippocampus, PND1 (MG-ACTH = 48, MG-C = 37, LG-ACTH = 24, LG-C = 22), PND28 (MG-ACTH = 27, MG-C = 14, LG-ACTH = 40, LG-C = 32).
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06868
Areas of (A) adrenal cortex and (B) adrenal medulla in piglets on PND1 and PND28 from sows treated with ACTH (Synacthen Depot 100 IU i.m.; filled bars) or saline (open bars) during mid- and late gestation. *P < 0.05. Number of animals per group: adrenal cortex and medulla, PND1 (MG-ACTH = 28, MG-C = 25, LG-ACTH = 27, LG-C = 22), PND28 (MG-ACTH = 20, MG-C = 24, LG-ACTH = 40, LG-C = 32).
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06868
Number of cells per mm2 in the (A) adrenal cortex and (B) adrenal medulla of piglets on PND1 and PND28 from sows treated with ACTH (Synacthen Depot 100 IU i.m.; filled bars) or saline (open bars) during mid- and late gestation. *P < 0.05. Number of animals per group: adrenal cortex and medulla, PND1 (MG-ACTH = 28, MG-C = 25, LG-ACTH = 27, LG-C = 22), PND28 (MG-ACTH = 20, MG-C = 24, LG-ACTH = 40, LG-C = 32).
Citation: Journal of Endocrinology 191, 1; 10.1677/joe.1.06868
The authors thank the staff of the Behavioural Physiology Research Unit and the Experimental Pig Unit for their technical assistance. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Alexander SL & Irvine CH 1998 The effect of social stress on adrenal axis activity in horses: the importance of monitoring corticosteroid-binding globulin capacity. Journal of Endocrinology 157 425–432.
Andrews MH & Matthews SG 2000 Regulation of glucocorticoid receptor mRNA and heat shock protein 70 mRNA in the developing sheep brain. Brain Research 878 174–182.
Armario A, Giralt M, Mart O, Gavalda J, Hidalgo B, Hsu RS & Kuhn RW 1994 The effect of acute and chronic ACTH administration in pituitary–adrenal response to acute immobilization stress. Relationship to changes in corticosteroid-binding globulin. Endocrine Research 20 139–149.
Banjanin S, Kapoor A & Matthews SG 2004 Prenatal glucocorticoid exposure alters hypothalamic–pituitary–adrenal function and blood pressure in mature male guinea pigs. Journal of Physiology 558 305–318.
Barker DJP, Gluckman PD, Godfrey KM, Harding JE, Owens JA & Robinson JS 1993 Fetal nutrition and cardiovascular disease in adult life. Lancet 341 938–941.
Berridge CW & Foote SL 1991 Effects of locus coeruleus activation on electroencephalographic activity in neocortex and hippocampus. Journal of Neuroscience 11 3135–3145.
Bloom SL, Sheffield JS, McIntire DD & Leveno KJ 2001 Antenatal dexamethasone and decreased birth weight. Obstetrics and Gynecology 97 485–490.
Bowman RE, Maclusky NJ, Sarmiento Y, Frankfurt M, Gordon M & Luine VN 2004 Sexually dimorphic effects of prenatal stress on cognition, hormonal responses, and central neurotransmitters. Endocrinology 145 3778–3787.
Braastad BO 1998 Effects of prenatal stress on behaviour of offspring of laboratory and farmed animals. Applied Animal Behaviour Science 61 159–180.
Bright GM 1995 Corticosteroid-binding globulin influences kinetic parameters of plasma cortisol transport and clearance. Journal of Clinical Endocrinology and Metabolism 80 770–775.
Burlet G, Fernette B, Blanchard S, Angel E, Tankosic P, Maccari S & Burlet A 2005 Antenatal glucocorticoids blunt the functioning of the hypothalamic–pituitary–adrenal axis of neonates and disturb some behaviors in juveniles. Neuroscience 133 221–230.
Challis JRG, Matthews SG, Gibb W & Lye SJ 2000 Endocrine and paracrine regulation of birth at term and preterm. Endocrine Reviews 21 514–550.
Dean F & Matthews SG 1999 Maternal dexamethasone treatment in late gestation alters glucocorticoid and mineralocorticoid receptor mRNA in the fetal guinea pig. Brain Research 846 253–259.
Dent GW, Smith MA & Levine S 2000 Rapid induction of corticotropin-releasing hormone gene transcription in the paraventricular nucleus of the developing rat. Endocrinology 141 1593–1598.
Edwards CR, Benediktsson R, Lindsay RS & Seckl JR 1993 Dysfunction of the placental glucocorticoid barrier: link between the fetal environment and adult hypertension? Lancet 341 355–357.
Felix B, Leger ME & Albe-Fessard D 1999 Stereotaxic Atlas of the Pig Brain., Amsterdam: Elsevier Science BV.
Fiedler I, Rehfeldt C & Ender K 1996 Histophysiologische Kriterien der Schilddrüsen- und Nebennierenaktivität neugeborener Ferkel nach Behandlung der trächtigen Sau mit Somatotropin. Journal of Animal Physiology and Animal Nutrition 76 199–209.
Fowden AL, Li J & Forhead AJ 1998 Glucocorticoids and the preparation for life after birth: are there long-term consequences of the life insurance? Proceedings of the Nutrition Society 57 113–122.
Gerardin DCC, Pereira OCM, Kempinas WG, Florio JC, Moreira EG & Bernadi MM 2005 Sexual behavior, neuroendocrine, and neurochemical aspects in male rats exposed prenatally to stress. Physiology and Behavior 84 97–104.
Haussmann MF, Carroll JA, Weesner GD, Daniels MJ, Matteri RL & Lay DC 2000 Administration of ACTH to restrained, pregnant sows alters their pigs’ hypothalamic–pituitary–adrenal (HPA) axis. Journal of Animal Science 78 2399–2411.
Hayashi A, Nagaoka M, Yamadas K, Ichitani Y, Miake Y & Okado N 1998 Maternal stress induced synaptic loss and developmental disabilities of offspring. International Journal of Developmental Neuroscience 16 209–216.
Heo J, Kattesh HG, Roberts MP & Schneider JF 2003 Plasma levels of cortisol and corticosteroid-binding globulin (CBG) and hepatic CBG mRNA expression in pre- and postnatal pigs. Domestic Animal Endocrinology 25 263–273.
Heo J, Kattesh HG, Roberts MP, Morrow JL, Dailey JW & Saxton AM 2005 Hepatic corticosteroid-binding globulin (CBG) messenger RNA expression and plasma CBG concentrations in young pigs in response to heat and social stress. Journal of Animal Science 83 208–215.
Herrenkohl LR 1979 Prenatal stress reduces fertility and fecundity in female offspring. Science 206 1097–1099.
Jarvis S, Moinard C, Robson SK, Baxter E, Ormandy E, Douglas AJ, Seckl JR, Russell JA & Lawrence AB 2006 Programming the offspring of the pig by prenatal social stress: neuroendocrine activity and behaviour. Hormones and Behavior 49 68–80.
Kaehler ST, Singewald N, Sinner C, Thurnher C & Philippu A 2000 Conditioned fear and inescapable shock modify the release of serotonin in the locus coeruleus. Brain Research 859 249–254.
Kanitz E, Manteuffel G & Otten W 1998 Effects of weaning and restraint stress on glucocorticoid receptor binding capacity in limbic areas of domestic pigs. Brain Research 804 311–315.
Kanitz E, Tuchscherer M, Tuchscherer A, Stabenow B & Manteuffel G 2002 Neuroendocrine and immune responses to acute endotoxemia in suckling and weaned piglets. Biology of the Neonate 81 203–209.
Kanitz E, Otten W, Tuchscherer M & Manteuffel G 2003 Effects of prenatal stress on corticosteroid receptor binding and monoamine levels in limbic areas of neonatal pigs at different ages. Journal of Veterinary Medicine. Series A 50 132–139.
Kay G, Tarcic N, Poltyrev T & Weinstock M 1998 Prenatal stress depresses immune function in rats. Physiology and Behavior 63 397–402.
Kim CK, Yu W, Edin G, Ellis L, Osborn JA & Weinberg J 1999 Chronic intermittent stress does not differentially alter brain corticosteroid receptor densities in rats prenatally exposed to ethanol. Psychoneuroendocrinology 24 585–611.
Klemcke HG & Christenson RK 1997 Porcine fetal and maternal adrenocorticotropic hormone and corticosteroid concentrations during gestation and their relation to fetal size. Biology of Reproduction 57 99–106.
Konstandi M, Johnson E, Lang MA, Malamas M & Marselos M 2000 Noradrenaline, dopamine, serotonin: different effects of psychological stress on brain biogenic amines in mice and rats. Pharmacological Research 41 341–346.
Kranendonk G, Hopster H, Van Eerdenburg F, Van Reenen K, Fillerup M, De Groot J, Korte M & Taverne M 2005 Evaluation of oral administration of cortisol as a model for prenatal stress in pregnant sows. American Journal of Veterinary Research 66 780–790.
Kranendonk G, Hopster H, Fillerup M, Ekkel ED, Mulder EJH, Wiegant VM & Taverne MAM 2006 Lower birth weight and attenuated adrenocortical response to ACTH in offspring from sows that orally received cortisol during gestation. Domestic Animal Endocrinology 30 218–238.
Lay DC, Randel RD, Friend TH, Carroll JA, Welsh THJ, Jenkins OC, Neuendorff DA, Bushong DM & Kapp GM 1997a Effects of prenatal stress on fetal calf. Domestic Animal Endocrinology 14 73–80.
Lay DC, Randel RD, Friend TH, Jenkins OC, Neuendorff DA, Bushong DM, Lanier EK & Bjorge MK 1997b Effects of prenatal stress on suckling calves. Journal of Animal Science 75 3143–3151.
Lesage J, Del-Favero F, Leonhardt M, Louvart H, Maccari S, Vieau D & Darnaudery M 2004 Prenatal stress induces intrauterine growth restriction and programmes glucose intolerance and feeding behaviour disturbances in the aged rat. Journal of Endocrinology 181 291–296.
Levitt NS, Lindsay RS, Holmes MC & Seckl JR 1996 Dexamethasone in the last week of pregnancy attenuates hippocampal glucocorticoid receptor gene expression and elevates blood pressure in the adult offspring in the rat. Neuroendocrinology 64 412–418.
Levitt NS, Lambert EV, Woods D, Hales CN, Andrew R & Seckl JR 2000 Impaired glucose tolerance and elevated blood pressure in low birth weight, nonobese, young South African adults: early programming of cortisol axis. Journal of Clinical Endocrinology and Metabolism 85 4611–4618.
Liu L, Li A & Matthews SG 2001 Maternal glucocorticoid treatment programs HPA regulation in adult offspring: sex-specific effects. American Journal of Physiology. Endocrinology and Metabolism 280 E729–E739.
Lowry OH, Rosebrough NJ, Farr AL & Randall RJ 1951 Protein measurement with Folin reagent. Journal of Biological Chemistry 193 265–275.
Maccari S, Piazza PV, Kabbaj M, Barbazanges A, Simon H & Le Moal M 1995 Adoption reverses the long-term impairment in glucocorticoid feedback induced by prenatal stress. Journal of Neuroscience 15 110–116.
Maccari S, Darnaudery M, Morley-Fletcher S, Zuena AR, Cinque C & Van Reeth O 2003 Prenatal stress and long-term consequences: implications of glucocorticoid hormones. Neuroscience and Biobehavioral Reviews 27 119–127.
McPherson RL, Ji F, Wu G, Blanton JR & Kim SW 2004 Growth and compositional changes of fetal tissues in pigs. Journal of Animal Science 82 2534–2540.
Matthews SG 1998 Dynamic changes in glucocorticoid and mineralocorticoid receptor mRNA in the developing guinea pig brain. Developmental Brain Research 107 123–132.
Matthews SG 2002 Early programming of the hypothalamus–pituitary–adrenal axis. Trends in Endocrinology and Metabolism 13 373–380.
Moss TJ, Sloboda DM, Gurrin LC, Harding R, Challis JR & Newnham JP 2001 Programming effects in sheep of prenatal growth restriction and glucocorticoid exposure. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 281 R960–R970.
Muneoka K, Mikuni M, Ogawa T, Kitera K, Kamei K, Takigawa M & Takahashi K 1997 Prenatal dexamethasone exposure alters brain monoamine metabolism and adrenocortical response in rat offspring. American Journal of Physiology 273 R1669–R1675.
Otten W, Puppe B, Stabenow B, Kanitz E, Schön PC, Brüssow KP & Nürnberg G 1997 Agonistic interactions and physiological reactions of top and bottom ranking pigs confronted with a familiar and unfamiliar group: preliminary results. Applied Animal Behaviour Science 55 79–90.
Otten W, Kanitz E, Tuchscherer M & Nürnberg G 2001 Effects of prenatal restraint stress on hypothalamic–pituitary–adrenocortical and sympatho-adrenomedullary axis in neonatal pigs. Animal Science 73 279–287.
Otten W, Kanitz E, Tuchscherer M, Schneider F & Brüssow KP 2004 Effects of adrenocorticotropin stimulation on cortisol dynamics of pregnant gilts and their fetuses: implications for prenatal stress studies. Theriogenology 61 1649–1659.
Phillips DIW, Barker DJP, Fall CHD, Seckl JR, Whorwood CB, Wood PJ & Walker BR 1998 Elevated plasma cortisol concentrations: a link between low birth weight and the insulin resistance syndrom? Journal of Clinical Endocrinology and Metabolism 83 757–760.
Pond WG, Boleman SL, Fiorotto ML, Ho H, Knabe DA, Mersmann HJ, Savell JW & Su DR 2000 Perinatal ontogeny of brain growth in the domestic pig. Proceedings of the Society for Experimental Biology and Medicine 223 102–108.
Roussel S, Hemsworth PH, Boissy A & Duvaux-Ponter C 2004 Effects of repeated stress during pregnancy in ewes on the behavioural and physiological responses to stressful events and birth weight of their offspring. Applied Animal Behavior Science 85 259–276.
Roussel S, Boissy A, Montigny D, Hemsworth PH & Duvaux-Ponter C 2005 Gender-specific effects of prenatal stress on emotional reactivity and stress physiology of goat kids. Hormones and Behavior 47 256–266.
Rudeen PK & Weinberg J 1993 Prenatal ethanol exposure: changes in regional brain catecholamine content following stress. Journal of Neuroendocrinology 61 1907–1915.
Scatchard G 1949 The attraction of proteins for small molecules and ions. Annals of the New York Academy of Sciences 51 660–672.
Schwerin M, Kanitz E, Tuchscherer M, Brüssow KP, Nürnberg G & Otten W 2005 Stress-related gene expression in brain and adrenal gland of porcine fetuses and neonates. Theriogenology 63 1220–1234.
Seckl JR 1998 Physiologic programming of the fetus. Clinics in Perinatology 25 939–964.
Siiteri PK, Murai JT, Hammond GL, Nisker JA, Raymoure WJ & Kuhn RW 1982 The serum transport of steroid hormones. Recent Progress in Hormone Research 38 457–510.
Sloboda DM, Moss TJ, Gurrin LC, Newnham JP & Challis JRG 2002 The effect of prenatal betamethasone administration on postnatal ovine hypothalamic–pituitary–adrenal function. Journal of Endocrinology 172 71–81.
Slotkin TA, Lappi SE, McCook EC, Tayyeb MI, Eylers JP & Seidler FJ 1992 Glucocorticoids and the development of neural function: effects of prenatal dexamethasone exposure on central noradrenergic activity. Biology of the Neonate 61 326–336.
Spencer RL, Miller AH, Moday H, McEwen BS, Blanchard RJ, Blanchard DC & Sakai RR 1996 Chronic social stress produces reductions in available splenic type II corticosteroid receptor binding and plasma corticosteroid binding globulin levels. Psychoneuroendocrinology 21 95–109.
Statistical Analysis System Institute 1999 SAS/STAT UserGuide, version 8. SAS Institute Cary NC.
Stefanski V 2000 Social stress in laboratory rats: hormonal responses and immune cell distribution. Psychoneuroendocrinology 21 95–109.
Szuran TF, Pliska V, Pokorny J & Welzl H 2000 Prenatal stress in rats: effects on plasma corticosterone, hippocampal glucocorticoid receptors, and maze performance. Physiology and Behavior 71 353–362.
Takahashi LK, Turner JG & Kalin NH 1992 Prenatal stress alters brain catecholaminergic activity and potentiates stress-induced behaviour in adult rats. Brain Research 574 131–137.
Walker CD, Akana SF, Cascio CS & Dallman MF 1990 Adrenalectomy in the neonate: adult-like adrenocortical system responses to both removal and replacement of corticosterone. Endocrinology 127 823–842.
Ward IL 1972 Prenatal stress feminizes and demasculinizes the behavior of males. Science 175 82–84.
Weinstock M, Matlina E, Maor GI, Rosen H & McEwen BS 1992 Prenatal stress selectively alters the reactivity of the hypothalamic–pituitary-system in the female rat. Brain Research 595 195–200.
Weinstock M, Poltyrev T, Schorer-Apelbaum D, Men D & McCarty R 1998 Effect of prenatal stress on plasma corticosterone and catecholamines in response to footshock in rats. Physiology and Behavior 64 439–444.
Welberg LA, Seckl JR & Holmes MC 2001 Prenatal glucocorticoid programming of brain corticosteroid receptors and corticotrophin-releasing hormone: possible implications for behaviour. Neuroscience 104 71–79.
